The normal growth and development of the heart requires a temporally and spatially distinct pattern of expression of many cardiac genes during the developmental stages of heart formation. Several genes are strongly expressed in the developing heart, and subsequently down regulated in the adult heart, while others exhibit the inverse pattern of expression (46,47,51,68,9). A subset of cardiac genes which are preferentially expressed in the embryonic heart can be selectively induced in the adult heart during the abnormal growth and development of cardiac hypertrophy (23). Although the transcription frequency which are responsible for mediating these changes in cardiac gene expression are unknown, the involvement of members of the helix-loop-helix (HLH) family of transcription factors in mesodermal development has been well documented (14,21,63). As cardiac muscle is mesodermal in origin, this family is a likely candidate for playing a role in the expression of cardiac genes, both during normal and abnormal development, when embryonic/fetal genes are induced. To investigate the possible role of this family in cardiac gene expression, the Specific Aims of this proposal are a follows. Specific Aim 1: To determine whether specific members of the HLH family are expressed in cardiac cells, and if expressed, to examine the temporal and spatial correlation between their expression and that of specific developmentally regulated cardiac genes. Specific Aim 2: To determine whether specific members of the HLH family are capable of regulating cardiac and skeletal muscle promoters in either non-cardiac or cardiac cell backgrounds. Specific Aim 3: To examine the role of specific members of the HLH family in the activation of embryonic/fetal gene expression in an in vitro model of ventricular cell hypertrophy. Specific Aim 4: To examine cardiac cell nuclear extracts for the presence of tissue specific protein complexes which recognize the HLH consensus target sequence CANNTG, or E-box. These studies will employ cultured myocardial cell models and a variety of molecular and cellular approaches, including Rnase protection, Northern blotting and in situ analyses,and co-transfection of HLH expression vectors with cardiac and skeletal promoter-luciferase reporter constructs. Gel mobility shift analysis will also be performed with cardiac extracts and HLH recognition sites. The results of these initial studies should clarify the role of HLH proteins in the regulation of cardiac gene expression during normal and abnormal cardiac development.